Bacterial microcompartments (metabolosomes) are closed polyhedral shells 100-150 nm diameter made of thin protein sheets (with pores less than 1 nm in diameter which can be positively or negatively charged), enclosing enzymes and cofactors for carbon fixation (carboxysomes) or various forms of fermentative metabolism. Although bacterial microcompartments were first seen over fifty years ago in photosynthetic cyanobacteria, their presence in the cytoplasm of heterotrophic bacteria was only confirmed in 1998, because they require induction by specific metabolites to form. In fact, twenty percent of bacterial genome sequences contain microcompartment structural genes, in many cases associated with enzymes of unknown function. A high percentage of bacteria therefore make a major investment in retaining and expressing large (20+ gene) operons encoding these structures and associated enzymes. It is believed the structures help bacterial metabolic efficiency by selective limitation of the shell pores on the passage of reactants, by metabolic channeling, or other mechanisms achieving temporary retention of small reaction intermediates within the structure, but these advantages have not been fully quantified.
Native microcompartment operons consist of a combination of genes specifying structural components making up the microcompartment shell, and genes specifying enzymes or cofactors, which are located within the microcompartment, or process products leaving the microcompartment. The diameter of identified pores in microcompartment shells of heterotrophic bacteria is sufficient to admit the typical primary substrates such as 1,2-propanediol, glycerol, or ethanolamine (Table 1). Most molecules consumed or produced by the natural enzyme-catalysed reactions within the microcompartment are less than 100 Daltons. The largest substrate molecules believed to enter and leave native microcompartments (on the basis of the location of the enzyme which utilises them) are cofactors such as Coenzyme A (MW 767.54 Da) which circulate between the microcompartment interior and the cytoplasm. Although they are present in commensal bacteria, expression of different microcompartment operons occurs in enteric pathogens in the intestine and after phagocytosis.
Recombinant microcompartments can be expressed heterologously in E. coli, both with and without the associated interior enzyme (Parsons, J., S. Frank, D. Bhella, M. Liang, M. B. Prentice, D. Mulvihill, and M. J. Warren, Synthesis of Empty Bacterial Microcompartments, Directed Organelle Protein Incorporation, and Evidence of Filament-Associated Organelle Movement. Molecular Cell, 2010. 38: p. 305-315). A clonable localisation signal comprising a short peptide enabling enzyme targeting to the microcompartment interior has been identified. Fusion of a 42 amino acid peptide (Parsons et al. 2010). or an 18 amino acid peptide (Fan, C., S. Cheng, Y. Liu, C. M. Escobar, C. S. Crowley, R. E. Jefferson, T. O. Yeates, and T. A. Bobik, Short N-terminal sequences package proteins into bacterial microcompartments. Proc Natl Acad Sci USA, 2010. 107(16): p. 7509-14) from the N-terminus of PduP enzymes localised green fluorescent protein within microcompartments. Multiple sequence alignment of microcompartment associated enzymes reveals conserved N-terminal extensions of approximately 18 amino acids compared with non-microcompartment associated homologues for diol dehydratase small and medium subunits (PduD,E) ethanolamine ammonia lyase small subunit Eut C, Eut G, and pyruvate formate lyase Pfl2 (Fan et al 2010). General features of these N-terminal extensions, incorporating conserved hydrophobic residues followed by a less conserved linker region have been described (Sutter, M., D. Boehringer, S. Gutmann, S. Gunther, D. Prangishvili, M. J. Loessner, K. O. Stetter, E. Weber-Ban, and N. Ban, Structural basis of enzyme encapsulation into a bacterial nanocompartment. Nat Struct Mol Biol, 2008. 15(9): p. 939-947). Compartmentalisation of the cellular interior is a functionally transforming process which underlies such radical events as the emergence of eukaryotes. Nanotechnological applications of biological compartment systems have included the use of viral capsids for DNA delivery and lumazine synthase enclosure of HIV protease (Worsdorfer, B., K. J. Woycechowsky, and D. Hilvert, Directed evolution of a protein container. Science, 2011. 331(6017): p. 589-92). Bacteria contain certain polymeric compounds often used as energy or nutrient stores, which are subject to dynamic synthesis and breakdown by different enzymes, according to prevailing conditions, usually under global regulatory control. One example is cyanophycin, an amino acid polymer originally detected in cyanobacteria and formed by the action of cyanophycin synthetase CphA, an enzyme which can be produced in recombinant form in E. coli (Aboulmagd, E., F. B. Oppermann-Sanio, and A. Steinbüchel, Molecular characterization of the cyanophycin synthetase from Synechocystis sp. strain PCC6308. Archives of Microbiology, 2000. 174(5): p. 297-306.)
Another example, polyphosphate kinase PPK1 (E.C. 2.7.4.1), forms inorganic polyphosphate polymers (metachromatic volutin granules) in bacterial cytoplasm by catalysing the reaction nATP(polyphosphate)n+nADP. This enzyme was the first enzyme (PPK) recognised to catalyse polyphosphate synthesis and is now usually termed PPK1 to differentiate it from another subsequently described group of enzymes (PPK2) which primarily catalyse GTP synthesis but also have limited phosphate polymerising capacity. Generally, any mentions of polyphosphate kinase or PPK without qualification in the literature and herein refer to PPK1. PPK1 is widely distributed in bacteria and some eukaryotes and is characterised by a highly conserved ATP-binding tunnel containing an autophosphorylating histidine residue (Zhu, Y., W. Huang, S. S. Lee, and W. Xu, Crystal structure of a polyphosphate kinase and its implications for polyphosphate synthesis. EMBO Rep, 2005. 6(7): p. 681-7). Dimerization is crucial for the enzymatic activity of PPK1. A PPK1 monomer includes four structural domains, the N-terminal (N) domain, the head (H) domain, and two related C-terminal (C1 and C2) domains. The ATP-binding tunnel is formed by N, C1, and C2 domains, so conserved residues required for ATP-binding are distributed throughout the length of all known PPK1 proteins. Although nATP←(polyphosphate)n+nADP is a reversible reaction, in E. coli this enzyme generally favours synthesis of polyphosphate over breakdown (Vmax ratio of 4.1). However, the balance between net accumulation and breakdown changes dynamically with growth phase, and external stimuli, in part due to the action of other enzymes.
E. coli also contains two exopolyphosphatases which release orthophosphate from the termini of long chain polyphosphate: (polyphosphate)n→(polyphosphate)n-1+Pi. These are PPX (E.C. 3.6.1.11, sometimes called PPX1) and its homologue guanosine pentaphosphate phosphohydrolase (GPPA or PPX2), both described by InterPro Accession IPR003695. GPPA (E.C. 3.6.1.40) also hydrolyses guanosine pentaphosphate (pppGpp) to guanosine tetraphosphate (ppGpp) with phosphate release, and pppGpp is a competitive inhibitor of PPX and the polyphosphate hydrolytic activity of GPPA. Amino acid starvation in E. coli leads to accumulation of large amounts of polyphosphate due to high levels of pppGpp produced in the stringent response inhibiting PPX.
Phosphate pollution in waterways and water treatment plants is a major problem. Removal of phosphate from wastewater is required to treat agricultural phosphate-containing discharges to reduce eutrophication, the algal blooms and “dead zones” in lakes and coastal marine ecosystems. The established biological method to remove inorganic phosphate from wastewater (Enhanced Biological Phosphate Removal, EBPR) relies on empirical selection by cyclical aerobic and anaerobic incubation of a community of uncultivated bacteria capable of temporary polyphosphate storage. There are various disadvantages to the process: the microbiological basis is not understood, it can take months of pumping to become established, and it is thereafter operationally unstable (prone to unexplained failure).
In medicine, oral phosphorus chelation therapy is required in the management of chronic renal failure subjects because of toxicity resulting from accumulation of dietary phosphate in the absence of urinary secretion (hyperphosphatemia). One method that was used for reducing phosphate in such subjects was the use of oral aluminum hydroxide. However, the use of aluminum hydroxide has severe side-effects due to build up of aluminum in the body. Calcium-based salts are an effective replacement for aluminum therapy and are currently the most widely used but there is a concern about their association with hypercalcaemia and vascular calcification. Newer oral phosphate binding medicaments with fewer side effects have recently been introduced in place of aluminum hydroxide and calcium-based salts, namely the anion exchange resin sevelamer hydrochloride, and lanthanum carbonate. However, these are both much more expensive than calcium or aluminium salts and financial considerations may adversely affect their provision for all renal patients requiring phosphate binding therapy.
It is an object of the present invention to overcome some of the above-mentioned problems.